Core Transcription Factors Research Articles

Triacylglycerol biosynthesis in shaded seeds of tung tree (Vernicia fordii) is regulated in part by Homeodomain Leucine Zipper 21.

Light quantity and quality affect many aspects of plant growth and development. However, few reports have addressed the molecular connections between seed oil accumulation and light conditions, especially dense shade. Shade-avoiding plants can redirect plant resources into extension growth at the expense of leaf and root expansion in an attempt to reach areas containing richer light. Here, we report that tung tree seed oil accumulation is suppressed by dense shade during the rapid oil accumulation phase. Transcriptome analysis confirmed that oil accumulation suppression due to dense shade was attributed to reduced expression of fatty acid and triacylglycerol biosynthesis-related genes. Through weighted gene co-expression network analysis, we identified 32 core transcription factors (TFs) specifically upregulated in densely shaded seeds during the rapid oil accumulation period. Among these, VfHB21, a class I homeodomain leucine zipper TF, was shown to suppress expression of FAD2 and FADX, two key genes related to α-eleostearic acid, by directly binding to HD-ZIP I/II motifs in their respective promoter regions. VfHB21 also binds to similar motifs in the promoters of VfWRI1 and VfDGAT2, two additional key seed lipid regulatory/biosynthetic genes. Functional conservation of HB21 during plant evolution was demonstrated by the fact that AtWRI1, AtSAD1, and AtFAD2 were downregulated in VfHB21-overexpressor lines of transgenic Arabidopsis, with concomitant seed oil reduction, and the fact that AtHB21 expression also was induced by shade. This study reveals some of the regulatory mechanisms that specifically control tung tree seed oil biosynthesis and more broadly regulate plant storage carbon partitioning in response to dense shade conditions.

Superenhancer-transcription factor regulatory network in malignant tumors.

ObjectiveThis study aims to identify superenhancer (SE)–transcriptional factor (TF) regulatory network related to eight common malignant tumors based on ChIP-seq data modified by histone H3K27ac in the enhancer region of the SRA database.MethodsH3K27ac ChIP-seq data of eight common malignant tumor samples were downloaded from the SRA database and subjected to comparison with the human reference genome hg19. TFs regulated by SEs were screened with HOMER software. Core regulatory circuitry (CRC) in malignant tumor samples was defined through CRCmapper software and validated by RNA-seq data in TCGA. The findings were substantiated in bladder cancer cell experiments.ResultsDifferent malignant tumors could be distinguished through the H3K27ac signal. After SE identification in eight common malignant tumor samples, 35 SE-regulated genes were defined as malignant tumor-specific. SE-regulated specific TFs effectively distinguished the types of malignant tumors. Finally, we obtained 60 CRC TFs, and SMAD3 exhibited a strong H3K27ac signal in eight common malignant tumor samples. In vitro experimental data verified the presence of a SE–TF regulatory network in bladder cancer, and SE–TF regulatory network enhanced the malignant phenotype of bladder cancer cells.ConclusionThe SE–TF regulatory network with SMAD3 as the core TF may participate in the carcinogenesis of malignant tumors.

Cancer CRC: A Comprehensive Cancer Core Transcriptional Regulatory Circuit Resource and Analysis Platform.

A core transcriptional regulatory circuit (CRC) is a group of interconnected auto-regulating transcription factors (TFs) that form loops and can be identified by super-enhancers (SEs). Studies have indicated that CRCs play an important role in defining cellular identity and determining cellular fate. Additionally, core TFs in CRCs are regulators of cell-type-specific transcriptional regulation. However, a global view of CRC properties across various cancer types has not been generated. Thus, we integrated paired cancer ATAC-seq and H3K27ac ChIP-seq data for specific cell lines to develop the Cancer CRC (http://bio.liclab.net/Cancer_crc/index.html). This platform documented 94,108 cancer CRCs, including 325 core TFs. The cancer CRC also provided the “SE active core TFs analysis” and “TF enrichment analysis” tools to identify potentially key TFs in cancer. In addition, we performed a comprehensive analysis of core TFs in various cancer types to reveal conserved and cancer-specific TFs.

DcWRKY75 promotes ethylene induced petal senescence in carnation (Dianthus caryophyllus L.).

Carnation (Dianthus caryophyllus L.) is one of the most important and typical ethylene sensitive cut flowers worldwide, although how ethylene influences the petal senescence process in carnation remains largely unknown. Here, we screened out one of the key transcription factors, DcWRKY75, using a constructed ethylene induced petal senescence transcriptome in carnation and found that it shows quick induction by ethylene treatment. Silencing of DcWRKY75 delays ethylene induced petal senescence in carnation. Molecular evidence confirms that DcWRKY75 can bind to the promoter regions of two main ethylene biosynthetic genes (DcACS1 and DcACO1) and a couple of senescence associated genes (DcSAG12 and DcSAG29) to activate their expression. Furthermore, we show that DcWRKY75 is a direct target gene of DcEIL3-1, which is a homolog of the ethylene signaling core transcription factor EIN3 in Arabidopsis. DcEIL3-1 can physically interact with DcWRKY75 and silencing of DcEIL3-1 also delays ethylene induced petal senescence in carnation and inhibits the ethylene induced expression of DcWRKY75 and its target genes. The present study demonstrates that the transcriptional regulation network is vitally important for ethylene induced petal senescence process in carnation and potentially in other ethylene sensitive cut flowers.

Integrated bioinformatics analysis of core regulatory elements involved in keloid formation

BackgroundKeloid is a benign fibro-proliferative dermal tumor formed by an abnormal scarring response to injury and characterized by excessive collagen accumulation and invasive growth. The mechanism of keloid formation has not been fully elucidated, especially during abnormal scarring. Here, we investigated the regulatory genes, micro-RNAs (miRNAs) and transcription factors (TFs) that influence keloid development by comparing keloid and normal scar as well as keloid and normal skin.MethodsGene expression profiles (GSE7890, GSE92566, GSE44270 and GSE3189) of 5 normal scar samples, 10 normal skin samples and 18 keloid samples from the Gene Expression Omnibus (GEO) database were interrogated. Differentially expressed genes (DEGs) were identified between keloid and normal skin samples as well as keloid and normal scar samples with R Project for Statistical Computing. Gene Ontology (GO) functional enrichment analysis was also performed with R software. DEG-associated protein–protein interaction (PPI) network was constructed by STRING, followed by module selection from the PPI network based on the MCODE analysis. Regulatory relationships between TF/miRNA and target genes were predicted with miRnet and cytoscape. Core regulatory genes were verified by RT-qPCR.ResultsWe identified 628 DEGs, of which 626 were up-regulated and 2 were down-regulated. Seven core genes [neuropeptide Y(NPY), 5-hydroxytryptamine receptor 1A(HTR1A), somatostatin (SST), adenylate cyclase 8 (ADCY8), neuromedin U receptor 1 (NMUR1), G protein subunit gamma 3 (GNG3), and G protein subunit gamma 13 (GNG13)] all belong to MCODE1 and were enriched in the “G protein coupled receptor signaling pathway” of the GO biological process category. Furthermore, nine core miRNAs (hsa-mir-124, hsa-let-7, hsa-mir-155, hsa-mir-26a, hsa-mir-941, hsa-mir-10b, hsa-mir-20, hsa-mir-31 and hsa-mir-372), and two core TFs (SP1 and TERT) were identified to play important roles in keloid formation. In the TF/miRNA-target gene network, both hsa-mir-372 and hsa-mir-20 had a regulatory effect on GNG13, ADCY8 was predicted to be target by hsa-mir-10b, and HTR1A and NPY were potentially by SP1. Furthermore, the expression of core regulatory genes (GNG13, ADCY8, HTR1A and NPY) was validated in clinical samples.ConclusionsGNG13, ADCY8, NPY and HTR1A may act as core genes in keloid formation and these core genes establish relationship with SP1 and miRNA (hsa-mir-372, hsa-mir-20, hsa-mir-10b), which may influence multiple signaling pathways in the pathogenesis of keloid.

Molecular Mechanisms Underlying Cell-Fate Specification and Cellular Diversity of the Trophoblast Lineage during Placental Morphogenesis in Mice

Abstract Placental morphogenesis is a highly dynamic process involving mutual recognition and interlacing between the trophoblast-uterus and ultimately the initiation of the maternal-fetal circulatory system. During placental morphogenesis in mice, the trophoblast lineage, which integrates maternal and fetal signaling, undergoes stage-specific changes in gene regulatory programs directing cellular proliferation and fate specification, generating diverse trophoblast subtypes. While accumulating evidence from studies on genetically engineered and mutant mice has revealed the involvement of cell-specific core transcription factors in certain key events during placental morphogenesis, the precise molecular mechanisms by which multipotent trophoblasts gradually differentiate into different subtypes are still largely unknown. In this review, we primarily focus on mutant mouse models with placental phenotypes to provide a comprehensive understanding of the molecular mechanisms underlying cell-fate specification and cellular diversity of the trophoblast lineage during the placental morphogenesis.

ESRRB Facilitates the Conversion of Trophoblast-Like Stem Cells From Induced Pluripotent Stem Cells by Directly Regulating CDX2.

Porcine-induced pluripotent stem cells (piPSCs) could serve as a great model system for human stem cell preclinical research. However, the pluripotency gene network of piPSCs, especially the function for the core transcription factor estrogen-related receptor beta (ESRRB), was poorly understood. Here, we constructed ESRRB-overexpressing piPSCs (ESRRB-piPSCs). Compared with the control piPSCs (CON-piPSCs), the ESRRB-piPSCs showed flat, monolayered colony morphology. Moreover, the ESRRB-piPSCs showed greater chimeric capacity into trophectoderm than CON-piPSCs. We found that ESRRB could directly regulate the expressions of trophoblast stem cell (TSC)-specific markers, including KRT8, KRT18 and CDX2, through binding to their promoter regions. Mutational analysis proved that the N-terminus zinc finger domain is indispensable for ESRRB to regulate the TSC markers. Furthermore, this regulation needs the participation of OCT4. Accordingly, the cooperation between ESRRB and OCT4 facilitates the conversion from pluripotent state to the trophoblast-like state. Our results demonstrated a unique and crucial role of ESRRB in determining piPSCs fate, and shed new light on the molecular mechanism underlying the segregation of embryonic and extra-embryonic lineages.

DOC-2/DAB2 interactive protein destabilizes c-Myc to impair the growth and self-renewal of colon tumor-repopulating cells.

Colorectal carcinoma (CRC) remains a huge challenge in clinical treatment due to tumor metastasis and recurrence. Stem cell‐like colon tumor‐repopulating cells (TRCs) are a subpopulation of cancer cells with highly tumorigenic and chemotherapy resistant properties. The core transcription factor c‐Myc is essential for maintaining cancer stem‐like cell phenotypes, yet its roles and regulatory mechanisms remain unclear in colon TRCs. We report that elevated c‐Myc protein supported formation and growth of TRC spheroids. The tumor suppressor DOC‐2/DAB2 interactive protein (DAB2IP) suppressed c‐Myc expression to inhibit TRC expansion and self‐renewal. Particularly, DAB2IP disrupted c‐Myc stability through glycogen synthase kinase 3β/protein phosphatase 2A‐B56α‐mediated phosphorylation and dephosphorylation cascade on c‐Myc protein, leading to its eventual degradation through the ubiquitin‐proteasome pathway. The expression of DAB2IP was negatively correlated with c‐Myc in CRC specimens. Overall, our results improved mechanistic insight into how DAB2IP suppressed TRC growth and self‐renewal.

In This Issue

In This Issue

Abstract 39: Fate Of Renin Cell Progenitors In The Developing Kidney Vasculature

The unique spatial arregement of the kidney arterioles is an essential event for its development. However, the mechanisms that govern this process are still poorly understood. During nephrogenesis, a group of stromal cells expressing the Forkhead Box D1 ( FoxD1 ) transcription factor (TF) will give rise to the metanephric progenitors for the mural cells of the kidneys arteries and arterioles. We aim to identify the core TFs involved in the cell fate along the differentiaton pathways of the developing kidney vasculature. Therefore, we generated Foxd1-cre; mTmG mice, whose Foxd1 derivative cells are labeled with green fluorescent protein (GFP). GFP+ cells were isolated from 5 (P5) or 30 (P30) days old mice kidneys, and processed either for single-cell RNA-Seq (scRNA-Seq) or for single-cell Assay for Transposase-Accessible Chromatin (scATAC-Seq ). The top5 highly expressed TFs on scRNA-Seq at P5 are: Tcf21, Zeb2, Meis2, Cebpd and Nme3 (p_adjusted_value(padj)= 0, 3.8E-187, 3.9E-180, 4E-172, 4.1E-172 and 3.2E-154, respectively). They are involved in developmental processes and cell proliferation. At P30, the top5 highly expressed TFs are: Atf3, klf2, Fos, Nr4a2 and Junb (padj= 4.2E-294, 2.1E-200, 3.5E-182, 1.7E-52 and 0.2E-24, respectively). They are implicated with calcium-signaling pathway and inflammation. Additionally, scATAC-Seq identifies regions of accessible chromatin for pontential TFs binding, leading to changes in gene expression content and cell identity. At P30, scATAC-Seq showed differential accessible regions with subsequent putative motif enrichment analysis for the TF N4a2 (padj: 4E-297). This is in accordance with our scRNA-Seq results and might play a role in the Foxd1 progenitors cell fate decisions. Our results tracks the fate of the Foxd1+ cells during the kidney vasculature assembly and suggest a new transcription factors network that might play a role to orchestrate cell fate decisions during kidney vascular development.

The F-box E3 ubiquitin ligase BAF1 mediates the degradation of the brassinosteroid-activated transcription factor BES1 through selective autophagy in Arabidopsis.

Brassinosteroids (BRs) regulate plant growth, development, and stress responses by activating the core transcription factor BRI1-EMS-SUPPRESSOR1 (BES1), whose degradation occurs through the proteasome and autophagy pathways. The E3 ubiquitin ligase(s) that modify BES1 for autophagy-mediated degradation remain to be fully defined. Here, we identified an F-box family E3 ubiquitin ligase named BES1-ASSOCIATED F-BOX1 (BAF1) in Arabidopsis thaliana. BAF1 interacts with BES1 and mediates its ubiquitination and degradation. Our genetic data demonstrated that BAF1 inhibits BR signaling in a BES1-dependent manner. Moreover, BAF1 targets BES1 for autophagic degradation in a selective manner. BAF1-triggered selective autophagy of BES1 depends on the ubiquitin binding receptor DOMINANT SUPPRESSOR OF KAR2 (DSK2). Sucrose starvation-induced selective autophagy of BES1, but not bulk autophagy, was significantly compromised in baf1 mutant and BAF1-ΔF (BAF1 F-box decoy) overexpression plants, but clearly increased by BAF1 overexpression. The baf1 and BAF1-ΔF overexpression plants had increased BR-regulated growth but were sensitive to long-term sucrose starvation, while BAF1 overexpression plants had decreased BR-regulated growth but were highly tolerant of sucrose starvation. Our results not only established BAF1 as an E3 ubiquitin ligase that targets BES1 for degradation through selective autophagy pathway, but also revealed a mechanism for plants to reduce growth during sucrose starvation.

A Potential ceRNA Network for Neurological Damage in Preterm Infants.

Objective This study is aimed at identifying key genes involved in neurological damage in preterm infants and at determining their potential circRNA-miRNA-mRNA regulatory mechanisms. Methods Differentially expressed miRNAs, mRNAs, and circRNAs were downloaded from the GEO database. GO and KEGG enrichment analyses were used to determine possible relevant functions of differentially expressed mRNAs. The TTRUST database was used to predict differential TF-mRNA regulatory relationships. Then, CircMIR, miRDB, TargetScan and miRTarBase were then used to map circRNA/miRNA-TF/mRNA interaction networks. Finally, GSEA enrichment analysis was performed on the core transcription factors. Results A total of 640 mRNAs, 139 circRNAs, and 206 differentially expressed miRNAs associated with neurological injury in preterm infants were obtained. Based on the findings of Cytoscape and PPI network analysis, the hsa_circ_0008439-hsa-mir-3665-STAT3-MMP3 regulatory axis was established. GSEA analysis revealed that suppressed expression levels of STAT3 were associated with upregulated oxidative phosphorylation pathways in the neurological injury group of preterm infants. Conclusions The circRNA-miRNA-TF-mRNA regulatory network of neurological injury in preterm infants can be used to elucidate on the pathogenesis of brain injury and help us with the early detection of brain injury in preterm infants.

Integrated transcriptome and proteome analysis of developing embryo reveals the mechanisms underlying the high levels of oil accumulation in Carya cathayensis Sarg.

Hickory (Carya cathayensis Sarg.) is an extraordinary nut-bearing deciduous arbor with high content of oil in its embryo. However, the molecular mechanism underlying high oil accumulation is mostly unknown. Here, we reported that the lipid droplets and oil accumulation gradually increased with the embryo development and the oil content was up to ~76% at maturity. Furthermore, transcriptome and proteome analysis of developing hickory embryo identified 32,907 genes and 9857 proteins. Time-series analysis of gene expressions showed that these genes were divided into 12 clusters and lipid metabolism-related genes were enriched in Cluster 3, with the highest expression levels at 95days after pollination (S2). Differentially expressed genes and proteins indicated high correlation, and both were enriched in the lipid metabolism. Notably, the genes involved in biosynthesis, transport of fatty acid/lipid and lipid droplets formation had high expression levels at S2, while the expression levels of other genes required for suberin/wax/cutin biosynthesis and lipid degradation were very low at all the sampling time points, ultimately promoting the accumulation of oil. Quantitative reverse-transcription PCR analysis also verified the results of RNA-seq. The co-regulatory networks of lipid metabolism were further constructed and WRINKLED1 (WRI1) was a core transcriptional factor located in the nucleus. Of note, CcWRI1A/B could directly activate the expression of some genes (CcBCCP2A, CcBCCP2B, CcFATA and CcFAD3) required for fatty acid synthesis. These results provided in-depth evidence for revealing the molecular mechanism of high oil accumulation in hickory embryo.

A single nucleotide polymorphism in an R2R3 MYB transcription factor gene triggers the male sterility in soybean ms6 (Ames1)

Key messageIdentification and functional analysis of the male sterile gene MS6 in Glycine max.Soybean (Glycine max (L.) Merr.) is an important crop providing vegetable oil and protein. The male sterility-based hybrid breeding is a promising method for improving soybean yield to meet the globally growing demand. In this research, we identified a soybean genic male sterile locus, MS6, by combining the bulked segregant analysis sequencing method and the map-based cloning technology. MS6, highly expressed in anther, encodes an R2R3 MYB transcription factor (GmTDF1-1) that is homologous to Tapetal Development and Function 1, a key factor for anther development in Arabidopsis and rice. In male sterile ms6 (Ames1), the mutant allele contains a missense mutation, leading to the 76th leucine substituted by histidine in the DNA binding domain of GmTDF1-1. The expression of soybean MS6 under the control of the AtTDF1 promoter could rescue the male sterility of attdf1 but ms6 could not. Additionally, ms6 overexpression in wild-type Arabidopsis did not affect anther development. These results evidence that GmTDF1-1 is a functional TDF1 homolog and L76H disrupts its function. Notably, GmTDF1-1 shows 92% sequence identity with another soybean protein termed as GmTDF1-2, whose active expression also restored the fertility of attdf1. However, GmTDF1-2 is constitutively expressed at a very low level in soybean, and therefore, not able to compensate for the MS6 deficiency. Analysis of the TDF1-involved anther development regulatory pathway showed that expressions of the genes downstream of TDF1 are significantly suppressed in ms6, unveiling that GmTDF1-1 is a core transcription factor regulating soybean anther development.

BbWor1, a Regulator of Morphological Transition, Is Involved in Conidium-Hypha Switching, Blastospore Propagation, and Virulence in Beauveria bassiana.

ABSTRACTMorphological transition is an important adaptive mechanism in the host invasion process. Wor1 is a conserved fungal regulatory protein that controls the phenotypic switching and pathogenicity of Candida albicans. By modulating growth conditions, we simulated three models of Beauveria bassiana morphological transitions, including CTH (conidia to hyphae), HTC (hyphae to conidia), and BTB (blastospore to blastospore). Disruption of BbWor1 (an ortholog of Wor1) resulted in a distinct reduction in the time required for conidial germination (CTH), a significant increase in hyphal growth, and a decrease in the yield of conidia (HTC), indicating that BbWor1 positively controls conidium production and negatively regulates hyphal growth in conidium-hypha switching. Moreover, ΔBbWor1 prominently decreased blastospore yield, shortened the G0/G1 phase, and prolonged the G2/M phase under the BTB model. Importantly, BbWor1 contributed to conidium-hypha switching and blastospore propagation via different genetic pathways, and yeast one-hybrid testing demonstrated the necessity of BbWor1 to control the transcription of an allergen-like protein gene (BBA_02580) and a conidial wall protein gene (BBA_09998). Moreover, the dramatically weakened virulence of ΔBbWor1 was examined by immersion and injection methods. Our findings indicate that BbWor1 is a vital participant in morphological transition and pathogenicity in entomopathogenic fungi.IMPORTANCE As a well-known entomopathogenic fungus, Beauveria bassiana has a complex life cycle and involves transformations among single-cell conidia, blastospores, and filamentous hyphae. This study provides new insight into the regulation of the fungal cell morphological transitions by simulating three models. Our research identified BbWor1 as a core transcription factor of morphological differentiation that positively regulates the production of conidia and blastospores but negatively regulates hyphal growth. More importantly, BbWor1 affects fungal pathogenicity and the global transcription profiles within three models of growth stage transformation. The present study lays a foundation for the exploration of the transition mechanism of entomopathogenic fungi and provides material for the morphological study of fungi.

Inhibiting DNA-PK induces glioma stem cell differentiation and sensitizes glioblastoma to radiation in mice.

Glioblastoma (GBM), a lethal primary brain tumor, contains glioma stem cells (GSCs) that promote malignant progression and therapeutic resistance. SOX2 is a core transcription factor that maintains the properties of stem cells, including GSCs, but mechanisms associated with posttranslational SOX2 regulation in GSCs remain elusive. Here, we report that DNA-dependent protein kinase (DNA-PK) governs SOX2 stability through phosphorylation, resulting in GSC maintenance. Mass spectrometric analyses of SOX2-binding proteins showed that DNA-PK interacted with SOX2 in GSCs. The DNA-PK catalytic subunit (DNA-PKcs) was preferentially expressed in GSCs compared to matched non-stem cell tumor cells (NSTCs) isolated from patient-derived GBM xenografts. DNA-PKcs phosphorylated human SOX2 at S251, which stabilized SOX2 by preventing WWP2-mediated ubiquitination, thus promoting GSC maintenance. We then demonstrated that when the nuclear DNA of GSCs either in vitro or in GBM xenografts in mice was damaged by irradiation or treatment with etoposide, the DNA-PK complex dissociated from SOX2, which then interacted with WWP2, leading to SOX2 degradation and GSC differentiation. These results suggest that DNA-PKcs-mediated phosphorylation of S251 was critical for SOX2 stabilization and GSC maintenance. Pharmacological inhibition of DNA-PKcs with the DNA-PKcs inhibitor NU7441 reduced GSC tumorsphere formation in vitro and impaired growth of intracranial human GBM xenografts in mice as well as sensitized the GBM xenografts to radiotherapy. Our findings suggest that DNA-PK maintains GSCs in a stem cell state and that DNA damage triggers GSC differentiation through precise regulation of SOX2 stability, highlighting that DNA-PKcs has potential as a therapeutic target in glioblastoma.

Comparative analysis of the superoxide dismutase gene family in Cetartiodactyla.

Cetacea, whales, dolphins and porpoises form an order of mammals adapted to aquatic life. Their transition to an aquatic habitat resulted in exceptional protection against cellular insults, including oxidative and osmotic stress. Here, we considered the structure and molecular evolution of the superoxide dismutase (SOD) gene family, which encodes essential enzymes in the mammalian antioxidant system, in the superorder Cetartiodactyla. To this end, we juxtaposed cetaceans and their closest extant relatives (order Artiodactyla). We identified 94 genes in 23 species, of which 70 are bona fide intact genes. Although the SOD gene family is conserved in Cetartiodactyla, lineage-specific gene duplications and deletions were observed. Phylogenetic analyses show thatthe SOD2 subfamily diverged from a clade containing SOD1 and SOD3, suggesting that cytoplasmic, extracellular and mitochondrial SODs have started down independent evolutionary paths. Specific-amino acid changes (e.g. K130N in SOD2) that may enhance ROS elimination were identified in cetaceans. In silico analysis suggests that the core transcription factor repertoire of cetartiodactyl SOD genes may include Sp1, NF-κB, Nrf2 and AHR. Putative transcription factors binding sites responding to hypoxia were (e.g. Suppressor of Hairless; Su(H)) found in the cetaceanSOD1gene. We found significant evidence for positive selection in cetaceans using codon models. Cetaceans with different diving abilities also show divergent evolution of SOD1 and SOD2. Our genome-wide analysis ofSODgenes helps clarify their relationship and evolutionary trajectory and identify putative functional changes in cetaceans.

CD44 loss of function sensitizes AML cells to the BCL-2 inhibitor venetoclax by decreasing CXCL12-driven survival cues

AbstractAcute myeloid leukemia (AML) has a poor prognosis under the current standard of care. In recent years, venetoclax, a BCL-2 inhibitor, was approved to treat patients who are ineligible for intensive induction chemotherapy. However, complete remission rates with venetoclax-based therapies are hampered by minimal residual disease (MRD) in a proportion of patients, leading to relapse. MRD is a result of leukemic stem cells being retained in bone marrow protective environments; activation of the CXCL12-CXCR4 pathway was shown to be relevant to this process. An important role is also played by cell adhesion molecules such as CD44, which has been shown to be crucial for the development of AML. Here we show that CD44 is involved in CXCL12 promotion of resistance to venetoclax-induced apoptosis in human AML cell lines and AML patient samples, which could be abrogated by CD44 knock down, knockout, or blocking with an anti-CD44 antibody. Split-Venus bimolecular fluorescence complementation showed that CD44 and CXCR4 physically associate at the cell membrane upon CXCL12 induction. In the venetoclax-resistant OCI-AML3 cell line, CXCL12 promoted an increase in the proportion of cells expressing high levels of embryonic stem cell core transcription factors (ESC-TFs: Sox2, Oct4, Nanog) abrogated by CD44 knockdown. This ESC-TF–expressing subpopulation which could be selected by venetoclax treatment, exhibited a basally enhanced resistance to apoptosis and expressed higher levels of CD44. Finally, we developed a novel AML xenograft model in zebrafish, which showed that CD44 knockout sensitizes OCI-AML3 cells to venetoclax treatment in vivo. Our study shows that CD44 is a potential molecular target for sensitizing AML cells to venetoclax-based therapies.

A KMT2A-AFF1 gene regulatory network highlights the role of core transcription factors and reveals the regulatory logic of key downstream target genes.

Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than can be achieved by studying single TF binding events in isolation. Chromosomal translocations of the lysine methyltransferase 2A (KMT2A) gene produce KMT2A fusion proteins such as KMT2A-AFF1 (previously MLL-AF4), causing poor prognosis acute lymphoblastic leukemias (ALLs) that sometimes relapse as acute myeloid leukemias (AMLs). KMT2A-AFF1 drives leukemogenesis through direct binding and inducing the aberrant overexpression of key genes, such as the anti-apoptotic factor BCL2 and the proto-oncogene MYC. However, studying direct binding alone does not incorporate possible network-generated regulatory outputs, including the indirect induction of gene repression. To better understand the KMT2A-AFF1-driven regulatory landscape, we integrated ChIP-seq, patient RNA-seq, and CRISPR essentiality screens to generate a model GRN. This GRN identified several key transcription factors such as RUNX1 that regulate target genes downstream of KMT2A-AFF1 using feed-forward loop (FFL) and cascade motifs. A core set of nodes are present in both ALL and AML, and CRISPR screening revealed several factors that help mediate response to the drug venetoclax. Using our GRN, we then identified a KMT2A-AFF1:RUNX1 cascade that represses CASP9, as well as KMT2A-AFF1-driven FFLs that regulate BCL2 and MYC through combinatorial TF activity. This illustrates how our GRN can be used to better connect KMT2A-AFF1 behavior to downstream pathways that contribute to leukemogenesis, and potentially predict shifts in gene expression that mediate drug response.

EMBR-09. EXAMINING THE ROLE OF THE DEVELOPMENTALLY ENCODED TRANSCRIPTION FACTOR, LHX9, IN GROUP 3 MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Despite major advances in our understanding of the biology of MB, novel treatments remain urgently needed. Using a chemical-genomics driven drug repositioning strategy, we identified the cardiac glycoside family of compounds as potential treatments for Group 3 MB. We subsequently demonstrated that single-agent treatment with digoxin prolongs survival in a patient-derived xenograft model (PDOX) of Group 3 MB to a degree comparable to radiation therapy, a mainstay in the treatment of MB. Finally, we examined the mechanism of digoxin-mediated cell killing using RNA-seq. This work identified LHX9, a member of the LIM homeobox family of transcription factors, as the gene most significantly down-regulated following treatment (Huang and Injac et al, Sci Trans Medicine, 2018). Homologs of LHX9 play key roles in cerebellar development via spatially and temporally restricted expression and LHX9 has been proposed as a core transcription factor (TF) in the regulatory circuitry of Group 3 tumors. Loss of function of other core TFs has been shown to impact MB growth. The role of LHX9 in MB, however, has not been previously experimentally evaluated. We now report that knockdown of LHX9 in MB-derived cell lines results in marked growth inhibition. RNA-seq analysis of LHX9-depleted cells showed changes which included alterations in extracellular matrix-receptor interactions and TGFb signaling. These findings raise the possibility that loss of LHX9 plays a major role in digoxin-mediated cell killing and that LHX9 represents a key dependency required for the growth of Group 3 MB. Clinical targeting of core TFs would represent a novel approach to targeting this devastating disease.